The present invention provides compositions and method related to Borrelia burgdorferi toxins, in particular, the present invention provides methods and compositions for the diagnosis, treatment and prevention of Lyme disease.
Lyme disease is a potentially severe and complex multisystem disorder associated with the tick-borne spirochete Borrelia burgdorferi. The disease is transmitted to humans and other animals through arthropod bites. Indeed, with few exceptions (e.g., B. recurrentis and B. duttonii), all Borrelia species are maintained in nature by cycling through the wild animals (e.g., deer, rodents and fowl) and the ticks that feed upon them. Lyme disease was first officially recognized in North America in 1975, and has become recognized as the most prevalent tick-borne disease in the U.S. This recognition was due to an outbreak of disease in children in Lyme, Conn. The disease is now recognized as having a worldwide distribution, with cases recorded in may countries around the world. Lyme disease seems to be more severe than erythema chronicum migrans, a tick-borne syndrome reported in Europe, as early as 1908. The etiologic agent, B. burgdorferi remained unidentified until 1981, when it was isolated from Ixodes scapularis (I. dammini) from New York and later in the European tick vector I. ricinus (See, Schwan, et al., xe2x80x9cBorreliaxe2x80x9d in Murray, et al., [eds]. Manual of Clinical Microbiology, 6th ed., ASM Press, Washington, D.C., 1995, pp. 625-635). In addition to infection acquired through tick bites, infected tissues of mammalian reservoirs or patients may also transmit disease.
In the northeastern United States, Wisconsin and Minnesota, the deer tick, Ixodes dammini is the primary vector, while in the western United States and Europe, I. pacifious and I. ricinus are the main vectors, respectively. It is also possible that ticks of other genera may transmit the disease (See, Schwan, et al., xe2x80x9cBorreliaxe2x80x9d in Murray, et al., [eds]. Manual of Clinical Microbiology, 6th ed., ASM Press, Washington, D.C., 1995, pp. 625-635). The prevalence of infected ticks appears to vary widely among geographic regions, and is directly proportional to the reported number of lyme disease cases in a given location (See, e.g., Persing, et al., J. Clin. Microbiol., 1990, 28, 566-572).
Although there is not universal agreement, it appears that the Lyme disease spirochete, B. burgdoferi sensu lato, may be divided into three separate species, B. burdorferi sensu stricto, B. garinii, and B. afzelii. There appears to be no vector specificity among these proposed species, nor it is clear as to whether they cause identical diseases. However, preliminary studies conducted in Europe have indicated that different clinical manifestations may be associated with these three species (See, Schwan, et al., xe2x80x9cBorreliaxe2x80x9d in Murray, et al., [eds]. Manual of Clinical Microbiology, 6th ed., ASM Press, Washington, D.C., 1995, pp. 625-635).
The clinical symptoms of Lyme disease vary among individuals and during the time course of infection, and range from a relatively benign skin rash to severe arthritic, neurologic and cardiac manifestations. The most common clinical manifestation is the distinctive skin rash (xe2x80x9cerythema migrans,xe2x80x9d xe2x80x9cerythema chronicam migrans,xe2x80x9d or xe2x80x9cECMxe2x80x9d) which follows the bite of an infected tick. This rash is often accompanied by headache, stiff neck, myalgias, arthralgias, malaise, fatigue, and/or lymph node swelling. Weeks to months later, some infected patients develop meningoencephalitis, myocarditis, or migrating musculoskeletal pain. Even later in the course of disease, patients may experience intermittent attacks of oligoarticular arthritis or chronic arthritis in large joints, particularly in the knee. While Lyme disease appears to progress to dermatologic and neurologic manifestations more frequently in Europe, arthritis is a more common late manifestation observed in U.S. patients (See, Rose, et al., J. Clin. Microbiol., 1991, 29:524-432). Other clinical syndromes reported in Europe that may have the same etiologic agent include lymphocytoma (lymphadenosis benigna cutis), acrodermalitis chronica atrophicans, tick-borne meningoradiculitis (Garin-Bujadoux-Bannwarth""s syndrome), and myositis. Due to increased awareness and reporting, reported cases of Lyme disease have increased over time. Between 1982 and 1992, approximately 50,000 cases of Lyme disease were reported to the Centers for Disease Control (CDC), with 48 states reporting cases by 1992.
Treatment of Lyme disease typically consisting of oral antimicrobial therapy at the initial stages and high dosage intravenous antimicrobial therapy for the later manifestations. While treatment is often successful, symptoms sometimes persist or reappear after treatment, particularly during the later stages of disease (See, e.g., Rose and Schwan, J. Infect. Dis., 1989, 160:1018-1029). Chronic Lyme disease is difficult to treat with current antimicrobial regimes (Donta, et al., Clin. Infect. Dis., 1997, 25:552-555).
With the dramatic increase in public awareness of the disease, its prevalence and geographical distribution of Lyme borreliosis, a tremendous new demand has been placed on laboratories to confirm cases using methods such as serology (See, Simpson, et al., J. Clin. Microbiol., 1990, 28:1329-1337). However, diagnosis of Lyme disease is often not straightforward. For example, there are well-documented Lyme disease cases for which knowledge of tick bite, skin rash, or positive serology are lacking. Most of the systemic manifestations of advanced Lyme disease are not unique to the disease and observation of spirochetes in patients is difficult (See, Rosa and Schwan, J. Infect. Dis., 1989, 160:1018-1029; Rosa, et al., J. Clin. Microbiol., 1991, 29:524-532). In addition, strong, specific immunologic responses are not always observed.
Furthermore, many problems have been reported with currently available serological tests, including both false positives and false negatives. Some tests have focused on the flagellar protein of B. burgdorferi as a means to increase the sensitivity of the serological tests because early studies indicate that the 41 kD flagellar subunit (flagellin) generated the earliest immune response in infected humans (See, e.g., Simpson, et al., J. Clin. Microbiol., 1990, 28:1329-1337 for a brief review). However, potential factors have been considered problematic with the use of flagellar protein, including the cross-reactivity of antibodies to flagella of other Borrelia species, as well as other organisms such as Treponema pallidum. This has led others to investigate the use of a 39 kDa non-flagellar B. burgdoferi antigen that is immunoreactive with many sera from human Lyme disease patients (See, Simpson, et al., J. Clin. Microbiol., supra).
Additional factors complicate the serologic diagnosis of Lyme disease. For example, serology is often less useful during the early stages of disease (i.e., during primary Lyme disease), as many patients with ECM have not formed sufficient antibody levels to be detectable in many assays (See, e.g., Coleman and Benach, J. Infect. Dis., 1987, 155:756-765). Antigen detection in urine, blood, and other tissues by a dot blot immunoassay and an antigen capacity assay using colloidal gold-immune electron microscopy have also been tried. However, these antigen detection methods are apparently no longer available (See, Schwan, et al., xe2x80x9cBorrelia,xe2x80x9d in Murray, et al., [eds], Manual of Clinical Microbiology, 6th ed., ASM Press, Washington, D.C., 1995, pp.626-635).
In sum, much remains to be learned regarding Lyme disease and the pathogenic mechanisms associated with the disease. In addition, much remains to be learned regarding the organisms themselves. One complicated factor is the difficulty of culturing the organisms in the laboratory. Indeed, the maintenance of infectious spirochetes, requires the use of laboratory animals (e.g., rats, mice, or live colonies of infected ticks). Nonetheless, some investigations have provided useful information regarding the organism.
At lest one strain of B. burgdorferi (B31) has been reported to harbor a single linear chromosome of approximately one megabase pairs in size, making it one of the smallest bacterial genomes known (See, Casjens and Huang, Mol. Microbiol., 1993, 8:967-980). This chromosomal structure is a striking feature of these organisms, as no other bacterial groups are known to have linear chromosomes (Casjens and Huang, supra). These organisms are also somewhat unique among bacteria in that they carry numerous linear DNA molecules with covalently closed ends (xe2x80x9clinear plasmidsxe2x80x9d) (Simpson, et al., Infect. Immun., supra). The major outer membrane proteins xe2x80x9cOspAxe2x80x9d and xe2x80x9cOspBxe2x80x9d are encoded by a 49 kilobase pair (kb) linear plasmid in B. burgdorferi B31, and are carried on similarly-sized plasmids in other isolates.
Although the functions of OspA and OspB remains to be elucidated, it is possible that OspB plays a role in adherence. Although antibodies to these antigens have also been detected in the sera of Lyme disease patients, their use as serodiagnostic agents can be questioned due to their lack of antigenic stability.
A recombinant OspA vaccine, with a reported 70-80% preventative efficacy has been recently developed (See, Steere, et al., New Engl. J. Med, 1998, 339:209-215; Meurice, et al, Clin. Infect. Dis., 1997, 25 Suppl. 1:S71-75; and Parenti, et al, xe2x80x9cThe Safety and Efficacy of an Adjuvanted Lyme Disease Vaccine,xe2x80x9d in 11th International Scientific Conference on Lyme Disease and Other Spirochetal and Tick-Borne Disorders, 1998, New York), but it is uncertain whether it will prevent the establishment of infection and long-term sequelae. Indeed, it is possible that this vaccine will prove to be harmful to patients who have previously contracted Lyme disease.
Syphilis is a chronic systemic infection caused by Treponema pallidum, and is characterized by a primary lesion that appears following an incubation period of xcx9c3 weeks, and is usually associated with regional lymphadenopathy. A secondary bacteremic stage is associated with generalized mucocutaneous lesions and generalized lymphadenopathy, followed by a latent period of subclinical infection lasting many years. In about one third of untreated cases, the tertiary stage is characterized by progressive destructive mucocutaneous, musculoskeletal or parenchymal lesions, aortitis, or symptomatic central nervous disease.
In view of debilitating nature of the advanced stages of the disease, compositions and methods for accurate diagnosis, as well as methods and compositions for treatment and disease prevention remain critical. However, these needs have not previously been satisfactorily met.
The present invention provides compositions and methods related to Borrelia burgdorferi and Treponema pallidum toxins. In particular, the present invention provides methods and compositions for the diagnosis, treatment and prevention of Lyme disease (Borrelia burgdorferi) and Syphilis (Treponema pallidum).
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated binding polypeptides and binding agents which bind such polypeptides, including antibodies. The foregoing can be used, inter alia, in the diagnosis or treatment of conditions characterized by the expression of a Borrelia burgdorferi toxin, Bbtox1, and by the expression of a Treponema pallidum toxin, Tptox1, nucleic acid or polypeptide.
According to one aspect of the invention, isolated nucleic acid molecules that code for a Bbtox1 and/or a Tptox1 polypeptide are provided and include: (a) nucleic acid molecules which hybridize under stringent conditions to a molecule selected from the group consisting of a nucleic acid of SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID NO:17, and which code for a Bbtox1 (SEQ ID NO:1, SEQ ID NO:3) and/or a Tptox1 (SEQ ID NO:17) polypeptide, (b) deletions, additions and substitutions of (a) which code for a respective Bbtox1 and/or Tptox 1 polypeptide, (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, or (d) full-length complements of (a), (b) or (c). In certain embodiments, the isolated nucleic acid molecule comprises nucleotides 1-957 of SEQ ID NO:1. In some embodiments the isolated nucleic acid molecules are those comprising nucleotides 1-957 of SEQ ID NO:3. In further embodiments, the isolated nucleic acid molecule comprises nucleotides 1-762 of SEQ ID NO:17. The isolated nucleic acid molecule also can comprise a molecule which encodes the polypeptide of SEQ ID NO:2 or the polypeptide of SEQ ID NO:4, and has Bbtox1 toxin activity. The isolated nucleic acid molecule can further comprise a molecule which encodes the polypeptide of SEQ ID NO:18 and has Tptox1 toxin activity.
The invention in another aspect provides an isolated nucleic acid molecule selected from the group consisting of (a) a unique fragment of nucleic acid molecule of SEQ ID NO:1, SEQ ID NO:3, and/or SEQ ID NO:17 (of sufficient length to represent a sequence unique within the human genome), (b) full-length complements of (a), provided that the fragment includes a sequence of contiguous nucleotides which is not identical to a sequence selected from the sequence group consisting of (1) sequences having the GenBank accession numbers of Table 1, (2) full-length complements of (1), and (3) fragments of (1) and (2).
In one embodiment, the sequence of contiguous nucleotides is selected from the group consisting of (1) at least two contiguous nucleotides nonidentical to the sequence group, (2) at least three contiguous nucleotides nonidentical to the sequence group, (3) at least four contiguous nucleotides nonidentical to the sequence group, (4) at least five contiguous nucleotides nonidentical to the sequence group, (5) at least six contiguous nucleotides nonidentical to the sequence group, (6) at least seven contiguous nucleotides nonidentical to the sequence group.
In another embodiment, the fragment has a size selected from the group consisting of at least: 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20, nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, 1000 nucleotides and every integer length therebetween.
According to another aspect, the invention provides expression vectors, and host cells transformed or transfected with such expression vectors, comprising the nucleic acid molecules described above.
According to another aspect of the invention, an isolated polypeptide is provided. The isolated polypeptide is encoded by the foregoing isolated nucleic acid molecules of the invention. In some embodiments, the isolated polypeptide is encoded by the nucleic acid of SEQ ID NO:1, giving rise to a polypeptide having the sequence of SEQ ID NO:2 that has Bbtox1 toxin activity. In certain embodiments, the isolated polypeptide is encoded by the nucleic acid of SEQ ID NO:3, giving rise to a polypeptide having the sequence of SEQ ID NO:4 that has Bbtox1 toxin activity. In further embodiments, the isolated polypeptide is encoded by the nucleic acid of SEQ ID NO:17, giving rise to a polypeptide having the sequence of SEQ ID NO:18 that has Tptox1 toxin activity. In other embodiments, the isolated polypeptide may be a fragment or variant of the foregoing of sufficient length to represent a sequence unique within the human genome, and identifying with a polypeptide that has Bbtox1 and/or Tptox1 toxin activity. In another embodiment, immunogenic fragments of the polypeptide molecules described above are provided.
According to another aspect of the invention, isolated binding polypeptides are provided which selectively bind a polypeptide and/or at least one antigen determinate on a polypeptide encoded by the foregoing isolated nucleic acid molecules of the invention. Preferably the isolated binding polypeptides selectively bind a polypeptide (and/or at least one antigen determinate on a polypeptide) which comprises the sequence of SEQ ID NO:2, SEQ ID NO:4, or fragments thereof, and that do not recognize (xe2x80x9ccross-reactxe2x80x9d) with epitopes from toxin polypeptides of V. cholerae, E. coli, B. perfussis, P. aeruginosa, T pallidum, and/or C. diptheriae. Additionally, the isolated binding polypeptides, preferably, selectively bind a polypeptide (and/or at least one antigen determinate on a polypeptide) which comprises the sequence of SEQ ID NO:18, or fragments thereof, and that do not recognize (xe2x80x9ccross-reactxe2x80x9d) with epitopes from toxin polypeptides of V. cholerae, E. coli, B. pertussis, P. aeruginosa, B. burgdorferi, and/or C. diptheriae. In preferred embodiments, the isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to the Bbtox1 polypeptide). The present invention encompasses polyclonal, as well as monoclonal antibodies.
The invention also contemplates kits comprising a package including assays for Bbtox1 epitopes, Bbtox1 nucleic acids, and/or Tptox1 epitopes, Tptox1 nucleic acids,and instructions, and optionally related materials such as controls, for example, a number, color chart, or an epitope of the expression product of the foregoing isolated nucleic acid molecules of the invention, for comparing the level of Bbtox1 and/or Tptox1 polypeptides, or Bbtox1 and/or Tptox1 nucleic acid forms in a test sample to the level in a control sample. This comparison can be used to assess in a subject a risk of developing Lyme disease (Bbtox1) and or Syphilis (Tptox1). The kits may also include assays for other known genes, and expression products thereof, associated with other infectious agents.
The present invention also provides a fusion protein(s) comprising a portion of Bbtox1 protein and a non-toxin protein sequence. In particularly preferred embodiments, the Bbtox1 protein comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4.
The present invention also provides a fusion protein(s) comprising a portion of Tptox1 protein and a non-toxin protein sequence. In particularly preferred embodiments, the Tptox1 protein comprises the sequence of SEQ ID NO:18.
According to another aspect of the invention, a method for determining the level of Bbtox1 expression in a sample is provided. The method involves measuring expression of Bbtox1 in a test sample, and comparing the measured expression of Bbtox1 in the test sample to a control, as a measure of the level of Bbtox1 expression. The control can be a negative control, or a quantitated control of Bbtox1 expression. In one embodiment, the test sample is obtained from a subject suspected of being infected with B. burgdorferi. In certain embodiments, the test sample is a B. burgdorferi culture or isolate. Expression of Bbtox1 in the test sample can be Bbtox1 mRNA expression and/or Bbtox1 polypeptide expression. In some embodiments, Bbtox1 mRNA expression can be measured using the Polymerase Chain Reaction and/or northern blotting. In certain embodiments, Bbtox1 polypeptide expression can be measured using monoclonal and/or polyclonal antisera to Bbtox1. In further embodiments, the test sample can be tissue or a biological fluid.
The present invention also provides methods for producing anti-Bbtox1 antibodies comprising, exposing an animal having immunocompetent cells to an immunogen comprising at least an antigenic portion of a Bbtox1 polypeptide under conditions such that immunocompetent cells produce antibodies directed against the antigenic portion of the Bbtox1 polypeptide. In one embodiment, the method further comprises the step of harvesting the antibodies. In an alternative embodiment, the method comprises the step of fusing the immunocompetent cells with an immortal cell line under conditions such that a hybridoma is produced. In yet another embodiment, the portion of Bbtox1 used as an immunogen to generate the antibodies is at least a portion of SEQ ID NO:2 or SEQ ID NO:4. In another embodiment, the fusion protein comprises at least a portion of the Bbtox1 protein.
The present invention provides methods for detecting Bbtox1 comprising: providing in any order, a sample suspected of containing Bbtox1, an antibody capable of specifically binding to at least a portion of the Bbtox1; mixing the samples and the antibody under conditions wherein the antibody can bind to the Bbtox1; and detecting the binding. In preferred embodiments of the methods, the sample comprises a B. burgdorferi culture or isolate. In other preferred embodiments, the sample is from a subject suspected of being infected with B. burgdorferi. The methods of the present invention encompass any method for detection.
The present invention also provides methods for detection of polynucleotides encoding at least a portion of Bbtox1 in a biological sample (such as biological fluid) comprising the steps of: a) hybridizing at least a portion of the polynucleotide sequence comprising at least fifteen nucleotides, which hybridizes under stringent conditions to at least a portion of the polynucleotide sequence selected from the group consisting of the DNA sequences set forth in SEQ ID NO:1, SEQ ID NO:3, to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding at least a portion of Bbtox1 the biological sample. In one alternative embodiment of the methods, the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.
The present invention also provides methods for detecting Bbtox1 comprising the steps of: a) providing a sample suspected of containing Bbtox1; and a control containing a quantitated Bbtox1; and b) comparing the test Bbtox1 in the sample with quantitated Bbtox1 in the control to determine the relative concentration of the test Bbtox1 in the sample. In addition, the methods may be conducted using any suitable means to determine the relative concentration of Bbtox1 in the test and control samples, including but not limited to the means selected from the group consisting of Western blot analysis, Northern blot analysis, Southern blot analysis, denaturing polyacrylamide gel eletrophoresis (e.g., SDS PAGE), reverse transcriptase-coupled polymerase chain reaction (RI-PCR), enzyme-linked immunosorbent assay (ELISA or ELA), radioimmunoassay (RIA), and fluorescent immunoassay (FIA). Thus, the methods may be conducted to determine the presence of Bbtox1 in the genome of the source of the test sample, or the expression of Bbtox1 (mRNA or protein), as well as detect the presence of abnormal or mutated Bbtox1 proteins or gene sequences in the test samples.
In one preferred embodiment, the presence of Bbtox1 is detected by immunochemical analysis. For example, the immunochemical analysis can comprise detecting the binding of an antibody specific for an epitope of Bbtox1 (e.g., at least a portion of the protein encoded by SEQ ID NO:1, SEQ ID NO:3). In another preferred embodiment of the method, the antibody comprises polyclonal antibodies, while in another preferred embodiment, the antibody comprises monoclonal antibodies.
According to another aspect of the invention, a method for determining the level of Tptox1 expression in a sample is provided. The method involves measuring expression of Tptox1 in a test sample, and comparing the measured expression of Tptox1 in the test sample to a control, as a measure of the level of Tptox1 expression. The control can be a negative control, or a quantitated control of Tptox1 expression. In one embodiment, the test sample is obtained from a subject suspected of being infected with T. pallidum. In certain embodiments, the test sample is a T. pallidum culture or isolate. Expression of Tptox1 in the test sample can be Tptox1 mRNA expression and/or Tptox1 polypeptide expression. In some embodiments, Tptox1 mRNA expression can be measured using the Polymerase Chain Reaction and/or northern blotting. In certain embodiments, Tptox1 polypeptide expression can be measured using monoclonal and/or polyclonal antisera to Tptox1. In further embodiments, the test sample can be tissue or a biological fluid.
The present invention also provides methods for producing anti-Tptox1 antibodies comprising, exposing an animal having immunocompetent cells to an immunogen comprising at least an antigenic portion of a Tptox1 polypeptide under conditions such that immunocompetent cells produce antibodies directed against the antigenic portion of the Tptox1 polypeptide. In one embodiment, the method further comprises the step of harvesting the antibodies. In an alternative embodiment, the method comprises the step of fusing the immunocompetent cells with an immortal cell line under conditions such that a hybridoma is produced. In yet another embodiment, the portion of Tptox1 used as an immunogen to generate the antibodies is at least a portion of SEQ ID NO:17. In another embodiment, the fusion protein comprises at least a portion of the Tptox1 protein (SEQ ID NO:18).
The present invention provides methods for detecting Tptox1 comprising: providing in any order, a sample suspected of containing Tptox1, an antibody capable of specifically binding to at least a portion of the Tptox1; mixing the samples and the antibody under conditions wherein the antibody can bind to the Tptox1; and detecting the binding. In preferred embodiments of the methods, the sample comprises a T. pallidum culture or isolate. In other preferred embodiments, the sample is from a subject suspected of being infected with T. pallidum. The methods of the present invention encompass any method for detection.
The present invention also provides methods for detection of polynucleotides encoding at least a portion of Tptox1 in a biological sample (such as biological fluid) comprising the steps of: a) hybridizing at least a portion of the polynucleotide sequence comprising at least fifteen nucleotides, which hybridizes under stringent conditions to at least a portion of the polynucleotide sequence of SEQ ID NO:17, to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding at least a portion of Tptox1 the biological sample. In one alternative embodiment of the methods, the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.
The present invention also provides methods for detecting Tptox1 comprising the steps of: a) providing a sample suspected of containing Tptox1; and a control containing a quantitated Tptox 1; and b) comparing the test Tptox 1 in the sample with quantitated Tptox1 in the control to determine the relative concentration of the test Tptox1 in the sample. In addition, the methods may be conducted using any suitable means to determine the relative concentration of Tptox1 in the test and control samples, and such means are as described above in relation to Bbtox1 detection.
In one preferred embodiment, the presence of Tptox1 is detected by immunochemical analysis. For example, the immunochemical analysis can comprise detecting the binding of an antibody specific for an epitope of Tptox1 (e.g., at least a portion of the protein encoded by SEQ ID NO:18). In another preferred embodiment of the method, the antibody comprises polyclonal antibodies, while in another preferred embodiment, the antibody comprises monoclonal antibodies.
In other preferred embodiments of the present invention, antibodies directed against full-length or fragments of Bbtox1 are used therapeutically. In one preferred embodiment, these antibodies are administered as passive immunization against the deleterious effects of infection with B. burgdorferi. 
In further preferred embodiments of the present invention, antibodies directed against full-length or fragments of Tptox1 are also used therapeutically. In one preferred embodiment, these antibodies are administered as passive immunization against the deleterious effects of infection with T. pallidum. 
The present invention further provides a vaccine preparation comprising inactivated (non-toxic) Bbtox1 protein (or Tptox1) protein (i.e., as a toxoid preparation). It is contemplated that such Bbtox1 (or Tptox1) toxoid preparations will be used alone, as well as in combination with other preparations suitable for vaccine use.
In an alternative embodiment, the present invention provides a vaccine comprising a fusion protein, said fusion protein comprising a non-toxin protein sequence and at least a portion of Bbtox1. In certain embodiments Bbtox1 is encoded by the nucleic acid of SEQ ID NO:1, 3, or fragments thereof. In some embodiments Tptox1 is encoded by the nucleic acid of SEQ ID NO:17, or fragments thereof. The vaccine may be a monovalent vaccine [i.e., containing only a Bbtox1 (or Tptox1) fusion protein], a bivalent vaccine [i.e., containing both a Bbtox1 (or Tptox1) fusion protein and one other component] or a trivalent or higher valency vaccine. In a preferred embodiment, the Bbtox1 fusion protein is combined with a fusion protein comprising a non-toxin protein sequence and at least a portion of Bbotxl. The present invention is not limited by the nature of the portion of the Bbtox1 selected. In another preferred embodiment, the Tptox1 fusion protein is combined with a fusion protein comprising a non-toxin protein sequence and at least a portion of Tptox1. The present invention is not limited by the nature of the portion of the Tptox1 selected. The present invention is also not limited by the nature of the non-toxin protein sequence employed. In a preferred embodiment, the non-toxin protein sequence comprises a poly-histidine tract. A number of alternative fusion tags or fusion partners are known in the art (e.g., MBP, GST, protein A, etc.) and may be employed for the generation of fusion proteins comprising vaccines. When a fusion partner (i.e., the non-toxin protein sequence) is employed for the production of a recombinant Bbtox1 (Tptox1) protein, the fusion partner may be removed from the recombinant Bbtox1 (Tptox1) protein if desired (i.e., prior to administration of the protein to a subject) using a variety of methods known to the art (e.g., digestion of fusion proteins containing Factor Xa or thrombin recognition sites with the appropriate enzyme). For example, a number of the pETH vectors provide an N-terminal his-tag followed by a Factor Xa cleavage site. In a preferred embodiment, the vaccine is substantially endotoxin-free.
The present invention is not limited by the method employed for the generation of vaccine comprising fusion proteins comprising a non-toxin protein sequence and at least a portion of Bbtox1. The fusion proteins may be produced by recombinant DNA means using either native or synthetic genes sequences expressed in a host cell. The present invention is also not limited to the production of vaccines using recombinant host cells; cell free in vitro transcription/translation systems may be employed for the expression of the nucleic acid constructs encoding the fusion proteins of the present invention. An example of such a cell-free system is the commercially available TnT(trademark) Coupled Reticulocyte Lysate System (Promega). Alternatively, the fusion proteins of the present invention may be generated by synthetic means (i.e., peptide synthesis).
According to another aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition comprises an isolated polypeptide encoded by the foregoing isolated nucleic acid molecules of the invention, in an immunogenically effective amount to induce antibody production in an immunocompetent subject against at least one antigenic portion of said isolated polypeptide, and a pharmaceutically acceptable carrier. In certain embodiments, the isolated polypeptide is a polypeptide selected from the group consisting of a polypeptide having a sequence of amino acids 1-319 of SEQ ID NO:2, a polypeptide having a sequence of amino acids 1-319 of SEQ ID NO:4, a polypeptide having a sequence of amino acids consisting of an immunogenic portion of the polypeptide of SEQ ID NO:2, a polypeptide having a sequence of amino acids consisting of an immunogenic portion of the polypeptide of SEQ ID NO:4, a polypeptide having a sequence of amino acids 1-254 of SEQ ID NO:18, and a polypeptide having a sequence of amino acids consisting of an immunogenic portion of the polypeptide of SEQ ID NO:18. In important embodiments, the isolated polypeptide is substantially endotoxin-free.
According to another aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition comprises a Bbtox1 vaccine in an immunogenically effective amount to induce antibody production in an immunocompetent subject against at least one Bbtox1 immunogenic portion of said vaccine, and a pharmaceutically acceptable carrier. In important embodiments, the Bbtox1 vaccine is substantially endotoxin-free.
According to a further aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition comprises a Bbtox1 binding agent in a pharmaceutically effective amount to inhibit Bbtox1 toxin activity, and a pharmaceutically acceptable carrier. In one embodiment, the Bbtox1 binding agent is an isolated polypeptide which binds selectively a polypeptide encoded by any of the foregoing isolated nucleic acid molecules of the invention, and in relation to SEQ ID NO:1or SEQ ID NO:3 (Bbtox1). In an important embodiment, the isolated binding polypeptide binds to a polypeptide having the sequence of amino acids of SEQ ID NO:2 or SEQ ID NO:4. Preferably, the isolated binding polypeptide is an antibody or an antibody fragment selected from the group consisting of a Fab fragment, a F(ab)2 fragment or a fragment including a CDR3 region selective for the polypeptide having the sequence of amino acids of SEQ ID NO:2 or SEQ ID NO:4, or of an antigenic portion thereof.
According to still another aspect of the invention, a method for inhibiting Bbtox1 activity in a subject, is provided. The method involves administering to a subject in need of such treatment a Bbtox1 binding agent in a pharmaceutically effective amount to inhibit Bbtox1 activity. Preferred Bbtox1 binding agents are as desdribed in the foregoing embodiments of the invention. In certain embodiments, the method further comprises co-administering an antibiotic and/or an antibacterial agent.
According to yet another aspect of the invention, a method for conferring Bbtox1 passive immunization in a subject is provided. The method involves administering to an immunocompetent subject in need of such treatment a Bbtox1 vaccine, in an immunogenically effective amount to induce antibody production in the subject against at least one Bbtox1 immunogenic portion of said vaccine. In one embodiment, the Bbtox1 vaccine comprises at least a portion of a Bbtox1 polypeptide. In some embodiments, the Bbtox1 vaccine comprises a fusion protein, said fusion protein comprising a non-toxin protein sequence and at least a portion of a Bbtox1 polypeptide. Preferred Bbtox1 polypeptides, non-toxin protein sequence of the fusion protein, and Bbtox1 vaccines are as described in any of the foregoing embodiments. In certain embodiments, the method further comprises co-administering an antibiotic and/or an antibacterial agent.
In further aspects, the invention provides Tptox1 compositions, methods of T. pallidum infection diagnosis, and treatment of diseases associated with T. pallidum infection (e.g., Syphilis), analogous to the foregoing teachings relating to Bbtox1 and Lyme disease.
The present invention further provides methods and compositions suitable for the identification of homologous toxins in organisms, including but not limited to other Borrelia species, Treponema species, and other spirochetes. In these embodiments, the primers and probes produced during the development of the present invention are used to identify proteins with sequence similarities to Bbtox1 and/or Tptox1. Based on these experiments, the function of these putative toxins may then be determined. It is also intended that the methods and compositions of the present invention will find use in molecular diagnostic procedures. For example, it is contemplated that the PCR methods of the present invention will find use in molecular diagnostics to identify additional strains of B. burgdorferi and other Borrelia, etc., capable of producing Bbtox1. It is further contemplated that the primers utilized in the development of the present invention (See, SEQ ID NOS: 19-23, and the amino acid sequences set forth in SEQ ID NOS:7-16) will find use in methods for amplification of nucleic acid present in samples suspected of containing B. burgdorferi and/or T. pallidum.